1. Field of the Invention
The present invention relates to a sensitive and simple optical detection scheme, for on-chip, high sensitivity universal (refractive index) solute detection for molded plastic microfluidic systems. This detector, based on micro-interferometry, allows for picoliter detection volumes and universal analyte detection, a task not previously possible in plastic microfluidic devices. The detection system has numerous applications, including universal/RI detection for CE (capillary electrophoresis), CEC (capillary electrochromatography) and FIA, physiometry, cell sorting/detection by scatter, ultra micro calorimetry, flow rate and temperature sensing. Because the detector has universal response the technique is also well suited to proteomics efforts where label free protein and DNA assays are needed. Thus, the invention allows the target molecules to be studied in their native state without additional chemical derivation.
2. Description of the Background Art
The ability to perform sensitive, universal, non-invasive, nano-scale sensing is imperative. Many applications exist, including flow or velocity sensing, universal solute detection, calorimetry, time resolved enthalpies and antigen-antibody binding, where non-invasive, sub-nano-volume detection is not only desired but essential. The area of miniaturized total analysis systems is driven by the need to analyze large numbers of samples quickly. For example, tens of millions of samples have been and must be analyzed by electrophoresis in order to map the human genome. Other areas where microfabricated instrumentation would decrease analysis time and accelerate progress include the high-throughput screening of combinatorial libraries to aid in drug discovery, the development of a library of genetic fingerprints for the members of the U.S. military and the screening of blood samples for infectious disease. Recently it has been shown that Surface Plasmon Resonance (SPR) technique can be applied, with limited success, to some of the aforementioned tasks. Even though SPR can be used for label free molecule detection, its drawbacks include the necessity of using expensive gold-plated substrates (problematic for plastics) and high quality optical prisms for surface illumination. It addition, it has a complicated flow-cell designs for sample delivery and only works when the solute is within nanometers of the gold surface.
Interferometry is among one of the most sensitive optical detection techniques known and as such, the most promising refractive index (universal) measurement techniques alternative to SPR when probing of nano-scale environments is necessary. One of the most promising techniques for on-chip interferometric detection is disclosed in Applicants"" copending application, U.S. patent application Ser. No. 09/519,860, filed Mar. 6, 2000. The ""860 application discloses an on-chip interferometric backscatter detector (OCIBD), which employs a channel of capillary dimensions that is preferably etched in a substrate for reception of a sample to be analyzed. A laser source generates an easy to align, simple optical train comprised of an unfocused laser beam that is incident on the etched channel for generating the backscattered light. The backscattered light comprises interference fringe patterns that result from the reflective and refractive interaction of the incident laser beam with the channel walls and the sample. These fringe patterns include a plurality of light bands whose positions shift as the refractive index of the sample is varied, either through compositional changes or through temperature changes, for example. A photodetector detects the backscattered light and converts it into intensity signals that vary as the positions of the light bands in the fringe patterns shift, and can thus be employed to determine the refractive index (RI), or an RI related characteristic property, of the sample. Preferably, the etched channel has a generally hemispherical cross sectional shape. A unique multi-pass optical configuration is inherently created by the channel characteristics, and is based on the interaction of the unfocused laser beam and the curved surface of the channel, that allows interferometric measurements in small volumes at high sensitivity.
One of the challenges encountered in the development of the OCIBD disclosed in the ""860 application lies in the fabrication of the micro-capillary channels in the substrate. In one preferred embodiment, the substrate is formed from glass or silica. There are several reasons why glass and silica-based substrates were initially chosen. First, etched channels in such substrates can be fabricated by modifying the well-known procedures used by the electronics industry to manufacture silicon chips. Second, these substrates are optically transparent allowing the use of previously developed detection methodology. Third, like fused silica capillaries used in capillary electrophoresis, these substrates have charged silanol groups on their surface and thus are capable of generating electroosmotic flow, the most common form of fluid transport in microfabricated devices. Fourth, these substrates possess high bulk resistivity and dielectric breakdown field strengths allowing the high electrical field strengths, commonly used in capillary electrophoresis, to be applied across micro-fabricated channels filled with a low-conductivity buffer. Fifth, there exists a large number of surface modification procedures commonly used in separation science that are easily adapted to planar chips made of glass, silica or silicon.
Although great success has been achieved with glass chips, there are several additional requirements or limitations involved with their use that make them less desirable than other material substrates. First, all fabrication steps must be carried out under clean room conditions adding considerable overhead cost to the production of microfabricated instrumentation. Second, the masks that are used in the photolithography process are very expensive. Third, these devices are typically sealed by high temperature annealing requiring temperatures of up to 1000xc2x0 C., a process which is not trivial, is labor intensive and often produces low device yields. Fourth and finally, glass, silica or silicon based microfluidic devices are brittle and fragile, making them hard to work with and less robust.
To address the limitations of using silica or glass substrates, the present invention provides an on-chip interferometric backscatter detector that makes use of plastic substrates in which rectangular channels are formed. Surprisingly, even though one might initially assume that a rectangular channel would not be the proper geometry to provide the requisite backscattered reflections to generate the desired fringe patterns, the inventors have discovered that the system of the ""860 application works equally well when the hemispherical etched channel is replaced by a rectangular channel formed in plastic.
The use of plastics is advantageous because they are considerably cheaper and require less fabrication time than glass/silica/silicon substrates. For example, the time required to design and produce a new device in plastic can be done in a week at a cost of $100-$200 while producing microchannels in glass or silica can take several months and cost as much as several thousand dollars. Other attributes of using plastics are that high geometrical aspect ratios (nearly vertical walls) are obtainable, little to no clean room time is required, high temperature sealing processes are not necessary, conventional detection methodologies are compatible, and the resulting devices are cheap enough to be considered disposable. The use of a rectangular channel also greatly simplifies fabrication of he device as compared to that of a semi cylindrical channel.
While any plastic material can be used to form the channel substrate, the substrate is most preferably formed from polydimethylsiloxane (PDMS). PDMS as a substrate has become popular in the last few years for several reasons. First, it is extremely economical and microfabricated structures can be produced quickly. Second, PDMS is optically transparent at visible wavelengths and low in background fluorescence making conventional detection methodologies compatible. Third, it is chemically and physically inert. Fourth, it is electrically insulating allowing electrophoresis systems to be fabricated.
The rectangular channels are preferably formed in the substrate using a molding or other suitable technique. The dimensions of the channels can be varied over a wide range, and are limited primarily by the width of the incident laser beam. In particular, to provide the best results, the laser beam diameter should be no smaller than the channel width so that the entire channel will be illuminated by the beam, and preferably should be slightly, e.g., 5%, larger. This will insure that the laser light reflected off of the walls of the channel will generate the desired interference fringe patterns, despite the less than optimum rectangular geometry of the channel walls. To provide an acceptable device resolution, the laser beam diameter should not be too large, and should thus be limited to approximately 2 mm. The depth of the channel is also preferably, though not necessarily, larger than its width, say by 50% or more. However, this does not affect device performance and is selected rather to further simplify fabrication of the channel.
In the preferred embodiment, an optional reference channel is also employed to improve the accuracy of the resulting sample measurements made with the OCIBD. The two channels are located in close proximity of each other and are illuminated by the laser beam, either simultaneously or sequentially (in near-real-time). By monitoring position changes for both of the resulting fringe patterns, it is possible to discriminate the desired RI signal generated by the sample from the background. These background interferences can be produced by gradients flowing through the channels and/or by environmental perturbations such as temperature and pressure changes. Using the sample and reference configuration also allows accurate temperature compensation. Implementation of the reference channel and successful compensation of RI gradients will make the OCIBD insensitive to RI changes other than those due to the target compound in the sample channel and will facilitate reaction dynamics to be monitored in real-time with flowing systems. Ultimately using a sample-reference approach to OCIBD will lead to substantially improved signal-to-noise ratio (S/N) for the OCIBD system.